“RNA interference”, “post-transcriptional gene silencing”, “quelling”—these different names describe similar effects that result from the overexpression or misexpression of transgenes, or from the deliberate introduction of double-stranded RNA into cells (reviewed in Fire, Trends Genet. 15: 358-363, 1999; Sharp, Genes Dev 13: 139-141, 1999; Hunter, Curr Biol 9: R440-R442, 1999; Baulcombe, Curr Biol 9: R599-R601, 1999; Vaucheret et al., Plant J 16: 651-659, 1998). The injection of double-stranded RNA into the nematode Caenorhabditis elegans, for example, acts systemically to cause the post-transcriptional depletion of the homologous endogenous RNA (Fire et al., Nature 391: 806-811, 1998; and Montgomery et al., PNAS 95: 15502-15507, 1998). RNA interference, commonly referred to as RNAi, offers a way of specifically and potently inactivating a cloned gene, and is proving a powerful tool for investigating gene function. Although the phenomenon is interesting in its own right; the mechanism has been rather mysterious, but recent research—for example that recently reported by Smardon et al., Curr Biol 10: 169-178, 2000—is beginning to shed light on the nature and evolution of the biological processes that underlie RNAi.
RNAi was discovered when researchers attempting to use the antisense RNA approach to inactivate a C. elegans gene found that injection of sense-strand RNA was actually as effective as the antisense RNA at inhibiting gene function (Guo et al., Cell 81: 611-620, 1995). Further investigation revealed that the active agent was modest amounts of double-stranded RNA that contaminate in vitro RNA preparations. Researchers quickly determined the ‘rules’ and effects of RNAi which have become the paradigm for thinking about the mechanism which mediates this affect. Exon sequences are required, whereas introns and promoter sequences, while ineffective, do not appear to compromise RNAi (though there may be gene-specific exceptions to this rule). RNAi acts systemically—injection into one tissue inhibits gene function in cells throughout the animal. The results of a variety of experiments, in C. elegans and other organisms, indicate that RNAi acts to destabilize cellular RNA after RNA processing.
The potency of RNAi inspired Timmons and Fire (Nature 395: 854, 1998) to do a simple experiment that produced an astonishing result. They fed to nematodes bacteria that had been engineered to express double-stranded RNA corresponding to the C. elegans unc-22 gene. Amazingly, these nematodes developed a phenotype similar to that of unc-22 mutants that was dependent on their food source. The ability to conditionally expose large numbers of nematodes to gene-specific double-stranded RNA formed the basis for a very powerful screen to select for RNAi-defective C. elegans mutants and then to identify the corresponding genes.
Double-stranded RNAs (dsRNAs) can provoke gene silencing in numerous in vitro contexts including Drosophila, Caenorhabditis elegans, planaria, hydra, trypanosomes, fungi and plants. However, the ability to recapitulate this phenomenon in higher eukaryotes, particularly mammalian cells, has not been accomplished in the art. Nor has the prior art demonstrated that this phenomena can be observed in cultured eukaryotic cells. Additionally, the ‘rules’ established by the prior art have taught that RNAi requires exon sequences, and thus constructs consisting of intronic or promoter sequences were not believed to be effective reagents in mediating RNAi. The present invention aims to address each of these deficiencies in the prior art and provides evidence both that RNAi can be observed in cultured eukaryotic cells and that RNAi constructs consisting of non-exon sequences can effectively repress gene expression.